Reverse transcription is the process by which a retrovirus such as HIV-1 converts its genetic material (single-stranded RNA) into a double-stranded DNA copy that is integrated into host chromosomal DNA. This process is complex and is catalyzed by the virion-associated enzyme, reverse transcriptase (RT). However, another viral protein, the nucleocapsid protein (NC), is also required for efficient and specific viral DNA synthesis. (A) We study the mechanistic basis for NC activity. HIV-1 NC is a small, basic nucleic acid binding protein with two zinc fingers, each containing the invariant CCHC zinc-coordinating motifs. It is a nucleic acid chaperone, i.e., it has the ability to catalyze conformational rearrangements that lead to the most thermodynamically stable nucleic acid structures. This property is critical for promoting the two strand transfer events that are needed for synthesis of full-length plus- and minus-strand viral DNA. In minus-strand transfer, the initial product of reverse transcription, (-) strong stop DNA, is translocated to the 3-prime end of viral RNA (termed acceptor RNA) in a reaction facilitated by base-pairing of the complementary repeat regions, which are present at the ends of the RNA and DNA partners. (i) Recent studies have focused on a comparison of the nucleic acid chaperone activities of NC and Gag (the precursor to viral structural proteins including NC), using the minus-strand transfer assay, a highly sensitive read-out for chaperone function. We report for the first time that Gag facilitates strand transfer, although not as efficiently as NC. Other experiments demonstrate that Gag chaperone activity resides in the NC domain. Surprisingly, unlike NC, high concentrations of Gag block the DNA elongation step in strand transfer. The ability of Gag to act as a roadblock to DNA polymerization represents a novel regulatory mechanism to prevent premature reverse transcription prior to protease cleavage of the precursor. (ii) We have also shown that NC duplex destabilization activity together with RNase H cleavage block mispriming by non-polypurine tract RNAs during initiation of plus-strand DNA synthesis. These findings demonstrate a previously unrecognized role for NC in selection of the correct primer and ensuring the fidelity of plus-strand initiation. (B) Our interest in host proteins that might affect HIV-1 reverse transcription has led us to investigate human APOBEC3G (A3G), a cellular cytidine deaminase with two zinc finger domains, which blocks HIV-1 reverse transcription and replication in the absence of the viral protein known as Vif. The antiviral effect has been shown to be largely deaminase-dependent, but there is also a deaminase-independent component. Previously, we succeeded in purifying catalytically active A3G, which allowed us to provide a comprehensive molecular analysis of its deaminase and nucleic acid binding activities. (i) Another focus of our A3G studies has been to elucidate the mechanism for A3G inhibition of reverse transcription. Using purified proteins, we have investigated the interplay between A3G, NC, and RT in reconstituted reactions representing individual steps in the reverse transcription pathway. We have reported that A3G does not affect the kinetics of NC-mediated annealing or the RNase H activity of RT. In sharp contrast, A3G significantly inhibits all RT-catalyzed elongation reactions with or without NC and without a requirement for A3G catalytic activity. Data from single-molecule DNA stretching analyses and fluorescence anisotropy support an unusual mechanism for deaminase-independent inhibition of reverse transcription that is determined by critical differences in the nucleic acid binding properties of A3G, NC, and RT. (ii) In current work, we have begun to study the human APOBEC3A (A3A) protein, which has only one zinc finger domain and is a potent inhibitor of retrotransposition by Line-1 and Alu non-LTR elements. We have expressed A3A in E. coli and have purified the protein from bacterial extracts. Efforts are underway to find conditions that will yield larger amounts of purified protein for detailed biochemical and structural analysis. An assay to measure Line-1 retrotransposition is being developed and construction of structure-guided A3A mutations for use in this assay is in progress. (C) Our laboratory has also been investigating the role of the HIV-1 capsid protein (CA) in early postentry events, a stage in the infectious process that is still not completely understood. Our previous studies illuminated the intimate connection between infectivity, proper core morphology, structural integrity of the CA protein, and the ability to undergo reverse transcription. (i) More recently, we performed a study to provide new information on the plasticity of CA: specifically, its ability to tolerate changes in N-terminal hydrophobic residues crucial for CA structure. When one of the CA mutants, W23F, was subjected to long-term passage, a second-site suppressor mutation, W23F/V26I was isolated that partially restored the wild-type phenotype. A structural model that accommodates the spatial changes induced by the W23F and V26I mutations can explain the suppressor phenotype. These findings are novel and demonstrate that despite the limits imposed on assembly of CA structure, HIV-1 is able to partially adapt to severe structural distortions in a major viral protein. (ii) In current work, we are investigating the effect of point mutations in the five residues comprising the linker region that connects the N- and C-terminal domains of CA. Although all of the mutants produce virus particles, only two have infectivity in a single-cycle replication assay. The lack of infectivity is correlated with the appearance of aberrant cores as observed by transmission electron microscopy, detergent sensitivity and instability of the cores, and dominant-negative inhibition of wild-type infectivity. The results obtained thus far indicate that the linker region of CA is essential for proper CA structure and function and contributes to modulation of core stability. Further characterization of these mutants is underway.